Antibody against nox1 polypeptide, method of diagnosing cancer with the use of nox1 gene and method of screening cancer growth inhibitor

ABSTRACT

The present invention provides a diagnostic method for cancer, a screening method for a cancer growth inhibitor, and a pharmaceutical composition used in cancer therapy using a Nox1 gene associated with a mutant Ras oncogene. More specifically, the present invention relates to: a composition for producing an antibody, comprising a polypeptide coded for a Nox1 gene, a homologue thereof, and their peptide fragments; an antibody against the polypeptide coded for a Nox1 gene; and a method for detecting the antibody or Nox1-expressing mRNA.

TECHNICAL FIELD

The present invention relates to a diagnostic method for cancer, ascreening method for a cancer growth inhibitor, and a pharmaceuticalcomposition based on a cancer-associated gene and a polypeptide codedfor the gene. More specifically, the present invention relates to adiagnostic method for cancer; a screening method for a cancer growthinhibitor; and a pharmaceutical composition used in cancer therapy,using a Nox1 gene associated with a mutant Ras oncogene, a polypeptidecoded for the gene, and an antibody specific to the polypeptide.

BACKGROUND ART

A Mutant Ras oncogene is heretofore known to exert great influence onmammalian cell carcinogenesis and its progression (Non-Patent Document1). For example, the mutant Ras oncogene is considered to cancerateanimal cells and aggravate the cancer via a Raf-MAPKK-MAPK pathway, oneof its principal downstream pathways (Non-Patent Document 2).

Based on these findings, there have been developed a method for tumorsuppressor gene therapy comprising administering an expression vectorgene comprising a gene coding for p94RB to a targeted cancer celldepleted of N-ras tumor suppressor (Patent Document 1), and a method ofidentifying a tumor suppressor gene by use of H-ras, K-ras, and N-rasoncogenes (Patent Document 2).

Recent research has reported that cells transformed with mutant Ras geneproduce reactive oxygen species (ROS) such as superoxide and H₂O₂(Non-Patent Document 3). Low-level intracellular ROS plays a role as asignal molecule in growth factor-induced cell growth (Non-PatentDocuments 4 and 5), suggesting that a rise in ROS generation associatedwith mutant Ras gene possibly causes the abnormal growth of animalcells.

However, reactive oxygen-producing enzymes that are involved incarcinogenesis, cancer promotion, and so on, caused by mutant Ras genehave heretofore been unknown.

-   (Patent Document 1) National Publication of International Patent    Application No. 1996-508166-   (Patent Document 2) National Publication of International Patent    Application No. 1998-504448-   (Non-Patent Document 1) Lowy, D. R. Annu. Rev. Biochem. 62, 851-891    (1993)-   (Non-Patent Document 2) McCormick, F. TCB. 9, M53-M56 (1999)-   (Non-Patent Document 3) Irani, K. et al. Science 275, 1649-1652    (1997)-   (Non-Patent Document 4) Sundaresan, M. et al. Science. 270, 296-299    (1995)-   (Non-Patent Document 5) Bae, Y. S. et al. J. Biol. Chem. 272,    217-221 (1997)

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a diagnostic method forcancer and a screening method for a cancer growth inhibitor; and apharmaceutical composition used in cancer therapy using a Nox-1 geneassociated with a mutant Ras oncogene, a polypeptide coded by the gene,and an antibody specific to the polypeptide.

The present inventors conducted studies and consequently gained findingsthat a mutant Ras oncogene remarkably increases the expression of Nox1(1, 2, and 3), a homologue of the catalytic subunit ofsuperoxide-generating NADPH oxidase, in an MAPKK-MAPK pathway, and thatsiRNA of the Nox1 gene effectively suppresses phenotypes of a mutant Rasgene such as cell adhesion-dependent growth, morphological changes, andtumor formulation in athymic mice. Based on the findings, the presentinventors attained the object and completed the present invention.

Namely, the present invention provides a composition for producing anantibody, comprising: (1) a polypeptide comprising the amino acidsequence of SEQ ID NO: 2; (2) a polypeptide having an amino acidsequence mutated from the amino acid sequence of SEQ ID NO: 2 by thesubstitution, deletion, addition, and/or insertion of one or more aminoacid residues and inducing the production of an antibody specific to thepolypeptide comprising the amino acid sequence of SEQ ID NO: 2; or (3) apolypeptide fragment having a partial sequence of the polypeptide of (1)or (2) and inducing the production of an antibody specific to thepolypeptide comprising the amino acid sequence of SEQ ID NO: 2.

Moreover, the present invention provides a method for producing anantibody specific to a polypeptide comprising the amino acid sequence ofSEQ ID NO: 2, comprising administering the composition for producing anantibody to a mammal.

Furthermore, the present invention provides an antibody specific to apolypeptide comprising the amino acid sequence of SEQ ID NO: 2.

Furthermore, the present invention provides a diagnostic method forcancer, comprising bringing the antibody specific to a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2 into contact with abiological sample.

Furthermore, the present invention provides a diagnostic composition forcancer and a pharmaceutical composition for cancer therapy, comprisingthe antibody.

Furthermore, the present invention provides a diagnostic method forcancer, characterized by detecting a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 1 or a fragment thereof. It ispreferred that the detection should be performed by detecting thepolynucleotide or the fragment thereof by polymerase chain reaction orreal-time quantitative polymerase chain reaction.

Furthermore, the present invention provides siRNA corresponding to apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or afragment thereof.

Furthermore, the present invention provides a pharmaceutical compositionfor cancer therapy, comprising: siRNA corresponding to a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 1 or a fragmentthereof; and siRNA corresponding to a polynucleotide comprising anucleotide sequence at positions from 71 to 1615 of SEQ ID NO: 1 or afragment thereof.

Furthermore, the present invention provides a pharmaceutical compositionfor cancer therapy, comprising a cell transformed with siRNAcorresponding to a polynucleotide comprising the nucleotide sequence ofSEQ ID NO: 1 or a fragment thereof; and siRNA corresponding to apolynucleotide comprising a nucleotide sequence at positions from 71 to1615 of SEQ ID NO: 1 or a fragment thereof.

Furthermore, the present invention provides a screening method for acancer cell growth inhibitor targeted to a Nox1 gene, comprising: (1)transfecting a cell with a Nox1 gene; bringing the transformed cell intocontact with a substance to be screened; and detecting the inactivationof Nox1 activity (primary screening); and (2) investigating whether ornot the Nox1-inactivating substance suppresses cancer cell growth(secondary screening), thereby detecting the expression of the Nox1 geneand the inactivation of Nox1 activity.

DEFINITION

The term “polypeptide” used herein means a primary structure of an aminoacid sequence, a polypeptide fragment with the primary structure, apolypeptide having biological activity accompanied with athree-dimensional structure, or a protein.

A “Nox1 polypeptide” used herein refers to a polypeptide coded for aNox1 gene and includes both full-length polypeptide and polypeptidefragment.

The term “homologue” used herein refers to a polypeptide having homologyin an amino acid sequence to a polypeptide or protein with a particularamino acid sequence and having biological activity or antigenicity incommon with the polypeptide or protein.

The term “siRNA” used herein means a short RNA fragment that suppressesthe expression of a particular gene or a double-stranded RNA moleculethat consists of the RNA fragment and its complementary strand.

A “mutant Ras gene” used herein refers to a human Ras gene in a mutatedstate that is involved in tumor formation. The mutant Ras gene is, forexample, a human Ras gene having a K-Ras mutation of glycine at the 12thcodon to aspartic acid or to valine or arginines, which is commonly seenin human pancreatic cancer, as well as a human Ras gene having K-Rasmutations occurring at the 12th codon and additionally at the 13th and61st codons, which are found in lung cancer.

A “partial sequence of an amino acid sequence” of the polypeptide usedherein refers to a sequence that comprises at least 8 or moreconsecutive amino acid residues contained in the amino acid sequence.

An “oligonucleotide” used herein refers to a nucleotide that typicallycomprises less than 100 bases and preferably comprises 6 to 99 bases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) is a graph showing a result of gene expression analysisconducted by real-time quantitative polymerase chain reaction in Example1;

FIGS. 1(b), (c), and (d) are, results of detecting Nox1 expression inExample 2 by conducting real-time quantitative polymerase chain reactionin the same way as in Example 1;

FIG. 2 is an electrophoretic image obtained in Example 4 by conductingPCR by use of M13F and 3.0Rev as primers to confirm a result oftransformation of cell lines stably transfected with RNAi(1), RNAi(3),or RNAi(5);

FIG. 3 shows photographs obtained in Example 4, wherein morphologicalchanges in which Nox1 gene is involved are compared among cells;

FIG. 4 is a graph showing a result of measuring the adhesion-dependentcell growth of cell lines exhibited by soft agar culture in Example 4 inorder to examine the transformation of cells by observinganchorage-independent growth capacity;

FIG. 5 is a graph showing a growth curve obtained in Example 4 bymeasuring the number of each of K-Ras-NRK/neg-1 (neg-1),K-Ras-NRK/RNAi(1)-7 (i(1)-7), K-Ras-NRK/RNAi(3)-19 (i(3)-19), andK-Ras-NRK/RNAi(5)-7 (i(5)-7) cells in a liquid medium;

FIG. 6 is a graph showing a result of quantifying a decrease in NBT inExample 5 by solubilizing treated cells and measuring absorbance at 510nm;

FIG. 7 is a graph showing in Example 6 that a GFP-rat Nox1 fusionpolypeptide is produced, that the coexpression of RNAi(1), RNAi (3), andRNAi(5) inhibits the production of a GFP-rat Nox1 fusion proteindose-dependently on their vectors (Left panel), and that the RNAiconstructs have high specificity to their target gene (right panel);

FIG. 8 is an electrophoretic image obtained in Example 6 by conductingRT-PCR to confirm that the mRNA expression of endogenous Nox1 isinhibited in each of cells, as compared to K-Ras-NRK and K-Ras-NRK/neg-1cells;

FIG. 9 is an image obtained in Example 7 by immunoblotting by the sameapproach as in Example 6, wherein the image demonstrates that siRNA useddoes not inhibit GFP or GFP-human Nox1 expression;

FIG. 10 is a photograph showing the influence of reactivation of Nox1once inhibited by siRNA on cells in terms of morphological changes inK-Ras-NRK cells in Example 7;

FIG. 11 is a graph showing the influence of reactivation of Nox1 onceinhibited by siRNA on cells in terms of cell growth rates;

FIG. 12 is a graph showing a result of investigating adhesion-dependentcell growth capacity by use of a soft agar culture method in order toexamine the influence of reactivation of Nox1 once inhibited by siRNA oncells by observing anchorage-independent growth capacity;

FIG. 13 is a graph showing a result of observing morphological changesin cells in order to investigate the influence of Nox1 gene expressionon transformation with mutant Ras in Example 9; and

FIG. 14 is a graph showing the influence of siRNA on tumor formationcaused by mutant Ras by observing tumor formation in a nude mouse over 1month and measuring the volume of the formed tumor in Example 9.

MOST PREFERABLE MODE FOR CARRYING OUT THE INVENTION

A composition for producing an antibody of the present inventioncomprises: (1) a polypeptide comprising the amino acid sequence of SEQID NO: 2; (2) a polypeptide having an amino acid sequence mutated fromthe amino acid sequence of SEQ ID NO: 2 by the substitution, deletion,addition, and/or insertion of one or more amino acid residues andinducing the production of an antibody specific to the polypeptidecomprising the amino acid sequence of SEQ ID NO: 2; or (3) a polypeptidefragment having a partial sequence of the polypeptide of (1) or (2) andinducing the production of an antibody specific to the polypeptidecomprising the amino acid sequence of SEQ ID NO: 2.

The composition for producing an antibody of the present invention maycontain a polypeptide having at least 80% homology, preferably 90%homology, particularly preferably 95% homology, to the amino acidsequence of SEQ ID NO: 2. The homology of amino acid sequences usedherein refers to identity (%) between a reference amino acid sequenceand an amino acid sequence compared therewith when the sequences arealigned. The homology can be calculated using default (initial setting)parameters in analytical software such as BLAST (J. Mol. Biol., 215, 403(1990)) and FASTA (Methods in Enzymology, 183, 63-69) that are standardprograms for performing the homology search of nucleotide sequences andamino acid sequences.

The polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is apolypeptide coded for a Nox1 gene. The carcinogenesis and cancerprogression of tissue to be examined can be screened by detecting theproduction of the polypeptide. Although an antibody manufactured withthe composition is specific to a human Nox1 polypeptide, the antibodycan also be used in the screening of the carcinogenesis and cancerprogression of non-human animals when having specificity to homologuesof the polypeptide produced by the non-human animals. Thus, thecomposition for producing an antibody can also be used for producing anantibody for detecting a human cancer cell and an antibody for screeningthe carcinogenesis and cancer progression of mammals capable ofproducing human Nox1 polypeptide homologues.

The partial sequence of the amino acid sequence of SEQ ID NO: 2 is asequence that comprises at least 8 or more consecutive amino acidresidues contained in the amino acid sequence and refers to any of thoseserving as an epitope that induces the production of an antibodyspecific to the polypeptide comprising the amino acid sequence of SEQ IDNO: 2. When the partial sequence is used as an epitope, it can be usedin combination with an appropriate carrier protein.

The polypeptide fragment of the present invention is any fragment of thepolypeptides (1) and (2) and induces the production of an antibodyspecific to the polypeptide comprising the amino acid sequence of SEQ IDNO: 2.

The length of the peptide fragment is not particularly limited andhowever, is typically 20 to 200 amino acid residues, preferably 20 to100 amino acid residues, more preferably 20 to 70 amino acid residues,in length.

In the present specification, the N-terminuses (amino terminuses) of thepolypeptide, the homologue, and the polypeptide fragment (hereinafter,abbreviated to the polypeptide, if necessary) are indicated in the left,and the C terminuses (carboxyl terminuses) thereof are indicated in theright, in accordance with the standard notation. The polypeptide and soon, of the present invention can have a carboxyl group (—COOH),carboxylate (—COO—), amide (—CONH₂), or ester (—COOR) at the C terminus.Examples of the side chain R of this ester include: C₁ to C₆ alkylgroups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl; C₃ to C₈cycloalkyl groups such as cyclopentyl and cyclohexyl; C₆ to C₁₂ arylgroups such as phenyl and α-naphthyl; and alkyl groups such as phenyl-C₁to -C₂ alkyl groups such as benzyl and phenethyl or α-naphthyl-C₁ to -C₂alkyl groups such as α-naphthylmethyl.

The polypeptide and so on, of the present invention also includes: thosein which an amino group of an N-terminal amino acid residue is protectedwith a protecting group such as a formyl group and an acetyl group;those in which an N-terminal glutamine residue generated in vivo bycleavage is converted to pyroglutamic acid; those in which a substituent(e.g., —OH, —SH, an amino group, an imidazole group, an indole group, ora guanidino group) on the side chain of an intramolecular amino acid isprotected with a suitable protecting group; or a conjugated protein suchas so-called glycoprotein bound with a sugar chain.

Moreover, the polypeptide and so on, of the present invention can beused as a physiologically acceptable inorganic or organic acid-additionsalt. Examples of the inorganic acid-addition salt include salts ofhydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid,and examples of the organic acid-addition salt include salts of aceticacid, formic acid, propionic acid, fumaric acid, maleic acid, succinicacid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid, and benzenesulfonic acid.

Namely, the composition for producing an antibody of the presentinvention comprises the polypeptide, the homologue thereof, and theirpeptide fragments, or their acid-addition salts. The composition mayoptionally contain ingredients such as a vehicle, a diluent, and anadjuvant.

The present invention provides a method of producing an antibodyspecific to the polypeptide comprising the amino acid sequence of SEQ IDNO: 2, the homologue thereof, or the peptide fragment by use of thecomposition for producing an antibody.

An antibody manufactured by the method of the present invention is notparticularly limited as long as it can be used according to the objectof the present invention. Examples of the antibody include a human/mousechimeric antibody, a humanized antibody, or a human antibody. In thiscontext, the humanized antibody refers to any of those containingseveral percent of a mouse-derived antibody in the whole antibody, whilethe human antibody refers to an antibody derived 100% from a human.Meanwhile, the chimeric human antibody refers to any of those containing10 to 20% of a mouse-derived antibody. The antibody may be any ofpolyclonal and monoclonal antibodies.

The monoclonal antibody of the present invention can be manufactured bythe following procedures: at first, the composition for producing anantibody is administered to non-human mammals. A complete Freund'sadjuvant or incomplete Freund's adjuvant may be administered forenhancing the ability of the mammals to produce antibodies. Theadministration is typically performed once every 2 to 6 weeks with atotal of approximately 2 to 10 doses. Examples of the non-human mammalsinclude monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats,and chickens, with mice and rats generally preferred.

An individual in which antibody production has been observed is selectedfrom the non-human mammals immunized with the composition. Two to Fivedays after final immunization, antibody-producing cells contained in thespleen or lymph node are collected from the individual and fused withmyeloma cells from animals of the same or different species to therebycreate monoclonal antibody-producing hybridomas. Antibody titer inantiserum is measured by allowing a labeled protein to react with theantiserum and then measuring the activity of the labeling agent boundwith the antibody. Cell fusion can be performed by a routine method suchas the method of Kohler and Milstein (Nature, 256, 495, 1975). Forexample, polyethylene glycol (PEG) or a Sendai virus can be used as afusion promoter.

Examples of the myeloma cells include non-human mammalian myeloma cellssuch as NS-1, P3U1, SP2/O, and AP-1, with P3U1 particularly preferred. Apreferable ratio of the number of the antibody-producing cells (spleencells) to the number of the myeloma cells, used in the fusion, isapproximately 1:1 to 20. The spleen and myeloma cells can be fused byincubation at 20 to 40° C., preferably 30 to 37° C., for 1 to 10minutes, with PEG (preferably PEG1000 to PEG6000) added at aconcentration of approximately 10 to 80%.

The monoclonal antibody-producing hybridomas can be screened by avariety of methods. Examples of the methods include: a method thatinvolves bringing a hybridoma culture supernatant into contact with asolid phase (e.g., a microplate) where an antigen is adsorbed eitherdirectly or in combination with a carrier and subsequently bringing asolution containing an anti-immunoglobulin antibody labeled with aradioactive substance, an enzyme, or the like, into contact therewith todetect the monoclonal antibody bound to the solid phase; and a methodthat involves bringing a hybridoma culture supernatant into contact witha solid phase where an anti-immunoglobulin antibody or the like isadsorbed and subsequently bringing a solution of a protein labeled witha radioactive substance, an enzyme, or the like, into contact therewithto detect the monoclonal antibody bound to the solid phase.

The monoclonal antibody-producing hybridomas can be selected by aroutine method. For example, a medium for animal cells supplemented withHAT (hypoxanthine-aminopterin-thymidine), an RPMI 1640 medium containing10 to 20% fetal bovine serum, a GIT medium containing 1 to 10% fetalbovine serum (Wako Pure Chemical Industries), or a serum-free medium forculturing hybridomas (SFM-101; Nissui Pharmaceutical) can be used. Aculture temperature is 20 to 40° C., and a culture time is typically 5days to 3 weeks. The culture can typically be performed under 5%carbonic acid gas. Antibody titer in the hybridoma culture supernatantcan be measured in the same way as in the above-described measurement ofantibody titer in antiserum.

The obtained monoclonal antibody can be separated and purified by aroutine method such as: an immunoglobulin separation and purificationmethod such as salting-out, alcohol precipitation, isoelectricprecipitation, and electrophoresis; an absorption and desorption methodwith an ion exchanger (DEAE); an ultracentrifugation method; a gelfiltration method; an antigen-bound solid phase; or a specificpurification method performed by collecting only an antibody with aprotein A active adsorbent and then dissociating the bond to obtain theantibody.

Alternatively, the polyclonal antibody of the present invention can bemanufactured by immunizing non-human mammals with the polypeptide and soon serving as an immunizing antigen or a complex of the peptide fragmentthereof with a carrier protein and then collecting anantibody-containing component such as serum therefrom, followed byantibody separation and purification.

The mixing ratio of the immunizing antigen to the carrier protein isdetermined so that the antibody can efficiently be raised against thehapten that has been crosslinked with the carrier and used inimmunization. For example, approximately 0.1 to 20 parts by weight,preferably approximately 1 to 5 parts by weight, of bovine serumalbumin, bovine thyroglobulin, or hemocyanin can be used with respect to1 part by weight of the hapten. For example, glutaraldehyde,carbodiimide, maleimide-activated ester, and activated ester reagentscontaining a thiol or dithiopyridyl group can be employed in thecoupling between the hapten and the carrier.

The complex antigen may be administered alone or in combination with acarrier or diluent, or alternatively, for enhancing the ability of themammals to produce antibodies, with a complete Freund's adjuvant orincomplete Freund's adjuvant. The administration is typically performedonce every 2 to 6 weeks with a total of approximately 3 to 10 doses. Thepolyclonal antibody is collected from the blood, ascites, yolk, or thelike, of the immunized mammal. Polyclonal antibody titer in antiserumcan be measured in the same way as in the above-described measurement ofantibody titer in antiserum. The polyclonal antibody can be separatedand purified in the same way as in the above-described separation andpurification of the monoclonal antibody.

A diagnostic kit for cancer of the present invention comprises thepolyclonal or monoclonal antibody thus obtained. The diagnostic kitoptionally contains wells for immune reaction, a staining agent, anenzyme-labeled antibody for detection, a cleansing liquid, an antibodydiluent, a sample diluent, an enzyme substrate, a diluent of an enzymesubstrate solution, and additional reagents.

A pharmaceutical composition comprising the antibody of the presentinvention suppresses the expression of a specific protein coded for aNox1 gene and as such, can be employed in the prevention, delayedprogression, and treatment of carcinogenesis.

The antibody can be administered orally or parenterally, and theparenteral administration includes local administration to tissue.

When the pharmaceutical composition of the present invention is orallyadministered, pharmaceutical ingredients such as carriers widely used,for example, fillers, expanders, binders, disintegrants, disintegrationinhibitors, buffers, tonicity agents, emulsifiers, dispersants,stabilizers, coating agents, surfactants, absorption promoters,humectants, wetting agents, adsorbents, lubricants, and excipients canbe used. Additives such as coloring agents, preservatives, perfumes,flavoring agents, and sweetening agents may optionally be used.

Concrete examples of the pharmaceutical ingredients include: excipientssuch as milk sugar, white sugar, sodium chloride, grape sugar, urea,starch, calcium carbonate, kaoline, crystalline cellulose, and silicicacid; binders such as water, ethanol, simple syrup, grape sugar fluids,starch fluids, gelatin solutions, carboxymethylcellulose, shellac,methylcellulose, potassium phosphate, and polyvinylpyrrolidone;disintegrants such as dried starch, sodium alginate, agar powder,laminaran powder, sodium hydrogencarbonate, calcium carbonate,polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate,stearic acid monoglyceride, starch, and milk sugar; disintegrationinhibitors such as white sugar, stearic acid, cacao butter, andhydrogenated oil; absorption promoters such as quaternary ammonium saltsand sodium lauryl sulfate; humectants such as glycerin and starch;adsorbents such as starch, milk sugar, kaolin, bentonite, and colloidalsilicic acid; and lubricants such as purified talc and stearate. Inaddition, the technique of a drug delivery system known in the art isoptionally adopted for each of the dosage forms to provide for thesustained release, local application (e.g., troches, buccals, andsublingual tablets), drug release control, conversion to an entericcoated form, conversion to a gastrolytic form, and so on, of thepharmaceutical composition.

When the pharmaceutical composition of the present invention isparenterally administered, the pharmaceutical composition can be used informs such as administration by injection such as instillation,intravenous injection, hypodermic injection, and intramuscularinjection, and endorectal administration with suppositories, forexample, suppositories made of fats and oils and water-solublesuppositories. The pharmaceutical composition in such a form can beprepared with ease by a routine method using a typical carrier in thepharmaceutical field.

The pharmaceutical composition comprising the antibody of the presentinvention can be administered to, for example, humans or other mammals(e.g., rats, mice, guinea pigs, rabbits, sheep, pigs, cattle, horses,cats, dogs, and monkeys). The amount of the pharmaceutical compositionadministered appropriately differs depending on the conditions ofobjects to be administered, administration routes, and so on. Forexample, when orally administered, the pharmaceutical composition isgenerally administered at approximately 10 to 4000 mg, preferablyapproximately 20 to 2000 mg, more preferably approximately 50 to 500 mg,per day for an adult patient weighing 60 kg. When parenterallyadministered, it is preferred that the siRNA, for example in the form ofinjection, should be administered through the vein at a dose ofapproximately 10 to 2000 mg, preferably approximately 20 to 1000 mg,more preferably approximately 50 to 500 mg, per day for an adult patientweighing 60 kg although the dose differs depending on objects to beadministered, the condition of liver cancer, and so on.

Cancer diagnosis can be performed by detecting and quantifying a Nox1gene in biological tissue or bodily fluids by immunochemical assaycomprising bringing the antibody of the present invention into contactwith a biological sample.

When this immunochemical assay is conducted, the antibody of the presentinvention is held in a carrier. Examples of the carrier used in theassay include: gel particles such as agarose gels (e.g., Sepharose 4Band Sepharose 6B; Pharmacia Fine Chemicals), dextran gels (e.g.,Sephadex G-75, Sephadex G-100, and Sephadex G-200; Pharmacia FineChemicals), polyacrylamide gels (Bio-Gel P-30, Bio-Gel P-60, and Bio-GelP-100; BioRad Laboratories), and cellulose particles (Avicel; AsahiKasei); ion-exchange cellulose such as diethylaminoethylcellulose andcarboxymethylcellulose; physical adsorbents such as glass, siliconestrips, and stainless steel-based resins; immunoassay plates (Nunc); andweakly basic anion-exchange resins (e.g., Amberlite IR-4B and DOWEX-3;Dow Chemical).

The antibody can be held in the carrier by a routine method such ascyanogen bromide and glutaraldehyde methods. In a more convenient way,the carrier may be adsorbed physically onto the surface of the antibody.Examples of a labeling agent to be bound to the antibody includeradioactive isotopes, enzymes, fluorescent substances, and luminescentsubstances, with enzymes preferably used.

Any of those being stable and having high specific activity ispreferable as the enzyme, and peroxidase, alkaline phosphatase,P-D-galactosidase, and glucose oxidase can be used, with peroxidaseparticularly preferred.

A sample to be tested in the immunochemical assay system of the presentinvention is a bodily fluid such as urine, serum, plasma, and spinalfluid, or an animal cell or a culture supernatant thereof. When theimmunochemical assay of the present invention is conducted, a substanceto be analyzed such as a Nox1 polypeptide to be measured is added to theantibody held in the carrier to perform antigen-antibody reaction. Then,the bound substance of an anti-Nox1 antibody and the labeling agent isfurther added thereto and reacted. The resulting reaction product issupplemented with the labeling agent (e.g., substrate of enzyme). Theenzyme activity of the reaction product can be determined by measuringthe absorbance or fluorescence intensity of a generated substance. Theseprocedures are performed in advance on a standard solution of a knownamount of the labeling agent (e.g., enzyme) to construct a standardcurve that plots the relationship of absorbance or fluorescenceintensity versus the amount of the Nox1 polypeptide or the like. Thisstandard curve is compared with the absorbance or fluorescence intensityobtained from the substance to be analyzed (sample to be tested)comprising an unknown amount of the Nox1 polypeptide to measure theamount of the Nox1 polypeptide in the substance to be analyzed.

Moreover, the present invention provides a diagnostic method for cancer,characterized by detecting a polynucleotide comprising the nucleotidesequence of SEQ ID NO: 1 or a fragment thereof. The polynucleotide orthe fragment thereof can be detected by a routine method utilizingpolymerase chain reaction (PCR) or real-time quantitative polymerasechain reaction.

The polymerase chain reaction or the real-time quantitative polymerasechain reaction uses a sense strand fragment corresponding to thenucleotide sequence of SEQ ID NO: 1 as a forward primer and an antisensestrand fragment corresponding to the nucleotide sequence of SEQ ID NO: 1as a reverse primer. The length of the forward primer dose not have tobe particularly limited and however, is typically 14 to 60 bases.Similarly, although the length of the reverse primer dose not have to beparticularly limited, it is usually preferred that the length should be14 to 60 bases. Examples of the forward and reverse primers used in thediagnostic method for cancer of the present invention include thefollowings: (SEQ ID NO: 5) the forward primer of5′-GGAGCAGGAATTGGGGTCAC-3′; and (SEQ ID NO: 6) the reverse primer of5′-TTGCTGTCCCATCCGGTGAG-3′.

When the detection is performed by the real-time quantitative polymerasechain reaction, examples of forward and reverse primers and a TaqManprobe used therein include the followings: the forward primer of (SEQ IDNO: 7) 5′-CCACTGTAGGCGCCCTAAGTT-3′; the reverse primer of (SEQ ID NO: 8)5′-AAGAATGACCGGTGCAAGGA-3′; and the TaqMan probe of (SEQ ID NO: 9)5′-AAGGGCATCCCCCTGAGTCTTGGAA-3′.

In the present invention, siRNA or siRNA corresponding to apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or afragment thereof suppresses Nox1 gene expression and reduces Nox1polypeptide production. Thus, the use of the siRNA or oligonucleotidewill allow the inhibition of cell carcinogenesis or cancer progressioninduced by a mutant Ras gene.

The siRNA or oligonucleotide of the present invention refers to anucleotide or an oligonucleotide complementary to a polynucleotidecomprising a consecutive nucleotide sequence of SEQ ID NO: 1 or afragment thereof.

The siRNA of the present invention may correspond to a polynucleotidecomprising a nucleotide sequence at positions from 71 to 1615 of SEQ IDNO: 1 or a fragment thereof. The length of the siRNA is less than 100 bpand is preferably 8 to 99 bp, particularly preferably 10 to 30 bp.Moreover, preferable examples of the ssi include the followings: siRNAsfor human Nox1 of (SEQ ID NO: 10)5′-GCGTGGCTTCAGCATGGAATTCAAGAGATTCCATGCTGAAGCCACGC TTTTTTGGAAA-3′; and(SEQ ID NO: 11) 5′-GGGCTTTCGAACAACAATATTCAAGAGATATTGTTGTTCGAAAGCCCTTTTTTGGAAA-3′; and siRNAs for rat Noxi of (SEQ ID NO: 12)5′-GTTATGAGAAGTCTGACAAGTTCAAGAGACTTGTCAGACTTCTCATA ATTTTTTGGAAA-3′; (SEQID NO: 13) 5′-GATTCTTGGCTAAATCCCATTCAAGAGATGGGATTTAGCCAAGAATCTTTTTTGGAAA-3′; and (SEQ ID NO: 14)5′-GGACATTTGAACAACAGCATTGAAGAGATGCTGTTGTTCAAATGTCC TTTTTTGGAAA-3′.

The siRNA of the present invention can be used by directlyadministrating the siRNA to a patient to be treated; by infecting thesiRNA into a cell taken out of a patient to be treated and thenreturning the cell to the patient; or by administering an expressionvector incorporating the siRNA therein to a patient to be treated andexpressing the siRNA.

Thus, the present invention provides a pharmaceutical composition forcancer therapy, comprising: siRNA corresponding to a polynucleotidecomprising the nucleotide sequence of SEQ ID NO: 1 or a fragmentthereof; and siRNA corresponding to a polynucleotide comprising anucleotide sequence at positions from 71 to 1615 of SEQ ID NO: 1 or afragment thereof.

The pharmaceutical composition can be manufactured as the foregoingpharmaceutical composition comprising the antibody, by a routine methodutilizing a pharmaceutical material conventionally used. Thepharmaceutical composition comprising the siRNA of the present inventioncan be administered to, for example, humans or other mammals (e.g.,rats, mice, guinea pigs, rabbits, sheep, pigs, cattle, horses, cats,dogs, and monkeys). The amount of the pharmaceutical compositionadministered appropriately differs depending on the conditions ofobjects to be administered, administration routes, and so on. Forexample, when orally administered, the pharmaceutical composition isgenerally administered at approximately 10 to 4000 mg, preferablyapproximately 20 to 2000 mg, more preferably approximately 50 to 500 mg,per day for an adult patient weighing 60 kg. When parenterallyadministered, it is preferred that the siRNA, for example in the form ofinjection, should be administered through the vein at a dose ofapproximately 10 to 2000 mg, preferably approximately 20 to 1000 mg,more preferably approximately 50 to 500 mg, per day for an adult patientweighing 60 kg although the dose differs depending on objects to beadministered, the condition of cancer to be treated, and so on.

Moreover, the present invention provides a pharmaceutical compositionfor cancer therapy, comprising a cell transformed with siRNAcorresponding to a polynucleotide comprising the nucleotide sequence ofSEQ ID NO: 1 or a fragment thereof; and siRNA corresponding to apolynucleotide comprising a nucleotide sequence at positions from 71 to1615 of SEQ ID NO: 1 or a fragment thereof.

In this context, the cell to be transformed may be a cell donated from athird party or a cell taken out of a patient to be treated.Alternatively, the transformed cell can be used as a cell for cancertherapy.

Moreover, the present invention provides an RNA molecule consisting of apartial sequence of the nucleotide sequence of SEQ ID NO: 1, or an RNAmolecule consisting of a mutant nucleotide sequence of the partialsequence with the addition, deletion, or substitution of at least onebase, and suppressing Nox1 polypeptide expression in a human cell. TheRNA molecule (siRNA) can be designed on the basis of the sequence of thepolynucleotide of the present invention according to a method known inthe art (e.g., Nature, Vol. 411, pp. 494, 2001).

When the RNA molecule of the present invention is used in vivo or invitro, it is used in the form of a double-stranded RNA molecule thatconsists of the RNA molecule and its complementary RNA. In this case, itis preferred that they should be treated so that the double-stranded RNAmolecule is not degraded in a cell or is not dissociated into singlestrands. The treatment is performed by, for example, a method that addsa hydroxyl group to the 3′ end of the RNA molecule, forms chemical bondsat both ends of the double strands through thiophosphoryl groups, orinduces a chemical bond between both strands by ultraviolet radiation orthrough a bifunctional group such as bis(2-chloromethyl)amine,4-thiouracil, or psoralen. The RNA molecule of the present inventionsuppresses Nox1 gene expression accompanying transformation with amutant Ras gene and as such, can be used as a pharmaceutical compositionfor the prevention of cell carcinogenesis, the inhibition of cancerprogression, and cancer therapy.

Moreover, the present invention provides a screening method for a cancercell growth inhibitor targeted for a Nox1 gene. The screening methodcomprises bringing a cell having a mutant Ras gene or a normal celltransfected with a Nox1 gene into contact with a substance to bescreened and then detecting the expression of the Nox1 gene and theinactivation of its enzyme activity. Subsequently, the transformed cellis cultured together with the substance to be screened to examine itsability to inhibit growth.

The influence of a compound to be screened on Nox1 gene expression canbe examined by detecting an increase or decrease in mRNA expression byreal-time quantitative polymerase chain reaction; by detecting apolypeptide or a peptide fragment thereof coded for the Nox1 gene withan antibody; or by observing morphological changes in the transformedcell.

The cell having a mutant Ras gene that can be used in the screeningmethod, is not particularly limited, and examples thereof includeH-Ras-NIH3T3 cells, K-Ras-NRK cells, and pancreatic cancer cells. Forexample, pEGFP-C1 (control) and pEGFP-C1-Nox1 can be used as a vectorused in the introduction of the Nox1 gene, while for example, pSilencercan be used as an siRNA-expressing vector.

A cell line highly expressing Nox1 can be established by stablytransfecting the pEGF-C1-Nox1 vector into an NIH3T3 or NRK cell by useof a Lipofectamine method. Alternatively, a pancreatic cancer cellhaving a mutant Ras gene can be utilized. It is preferred to use any ofthose separated and established from a human pancreatic cancer patientas the pancreatic cancer cell. These cells are cultured in a standardculture solution (Dulbecco's modified Eagle medium (DMEM) containing 10%fetal bovine serum (FBS)) in the presence of 5% CO₂. High-throughputscreening can be conducted by culturing the cells in a multi-plate (96or 48 wells), to which the substance to be screened is then added.

EXAMPLE 1 Confirmation of Nox1 Gene Expression Elevated by Mutant RasGene

Because Nox1 gene overexpression in NIH3T3 cells elevates superoxidegeneration and cell growth (1 and 2), the present inventors assumed thatNox1 is associated with the phenotypic expression of particularoncogenes. Therefore, cells transformed with K-Ras Val12 oncogene wereused to confirm Nox1 gene expression elevated by Ras oncogene.

At first, rat kidney (NRK) cells and K-Ras-NRK cells (Kirstein murinesarcoma virus-transformed NRK cells) were prepared. The K-Ras-NRK cellswere purchased from ATCC.

The presence or absence of Nox1 expression in the rat kidney (NRK) cellsand the K-Ras-NRK cells was initially detected by real-time quantitativepolymerase chain reaction. The real-time quantitative polymerase chainreaction used a forward primer of SEQ ID NO: 15, a reverse primer of SEQID NO: 16, and a TaqMan MGB probe of SEQ ID NO: 17. Forward Primer: (SEQID NO: 15) 5′-GGTCACTCCCTTTGCTTCCA-3′ Reverse Primer: (SEQ ID NO: 16)5′-GGCAAAGGCACCTGTCTCTCT-3′ TaqMan MGB Probe: (SEQ ID NO: 17)5′-TCCAGTAGAAATAGATCTTT-3′

The real-time quantitative polymerase chain reaction was performed usingABI Prism 7700 (Applied Biosystems) under reaction conditions set todefaults. Analytical values were standardized using rRNA 18S.

As a result of gene expression analysis by the real-time quantitativepolymerase chain reaction, Nox1 gene expression was elevated in theK-Ras-NRK cells transformed with the K-Ras oncogene, as shown in thegraph of FIG. 1(a).

EXAMPLE 2 Confirmation of Nox1 Gene Expression Elevated by TransientTransfection with Mutant Ras Gene

For analyzing the influence of transient transfection with mutant Rasgene, pcDNA3.1 vectors (blank vectors) and pcDNA3.1 carrying Ras Val12vectors were used and transiently transfected into NRK cells.Forty-eight hours after the transfection, real-time quantitativepolymerase chain reaction was conducted in the same way as in Example 1in order to detect Nox1 expression. As a result, Nox1 gene expressionwas also elevated in the NRK cells transiently transfected with theH-Ras Val12, as compared with Nox1 gene expression in the control vectorused, as shown in FIG. 1(b). The expression of the transfected Ras V-12was confirmed by immunoblotting (IB) using a rabbit anti-Ras antibody.

For performing the immunoblotting, 1×10⁵ sample cells were solubilizedwith 2× sample buffers (0.1 M Tris-Cl (pH 6.8), 20% glycerol, 4% SDS,3.1% DTT, and 0.001% BPB), and the polypeptides were separated by SDSgel electrophoresis and subsequently analyzed by the immunoblotting.

The proteins were electrically transferred to a nitrocellulose membranein a transfer buffer (25 mM Tris-Cl (pH 8.3), 92 mM glycine, and 20%methanol) and reacted with an anti-Ras antibody (primary antibody) andan HRP-conjugated anti-rabbit-IgG antibody, followed by detection by achemiluminescence (ECL) method.

As described above, the results of Examples 1 and 2 indicated that Nox1gene expression is elevated by the action of mutant Ras oncogene.

NIH3T3 cells cultured overnight under serum-free conditions were treatedmitogeniclally with serum (30%) or 50 ng/ml epidermal growth factor(EGF) and then analyzed by real-time quantitative polymerase chainreaction. As a result, Nox1 gene expression was increased to 6 to 20times within 12 hours from the initiation of the culture, as shown inFIGS. 1(c) and (d). That is, Nox1 gene expression is associated withfactors that promote cell growth. This is consistent with the reportssaying that both platelet-derived growth factor (PDGF) and angiotensinII induce superoxide formation and Nox1 gene expression in vascularsmooth muscle cells (1 and 4).

EXAMPLE 3 Analysis of Signaling Pathway of Nox1 Gene Expression Causedby Mutant Ras Gene

A series of experiments were performed for analyzing the downstreamregion of a Ras signaling pathway of Nox1 gene expression elevated bymutant Ras gene. In Examples 1 and 2, the cells were treated for 12hours with PD98059 (PD: 20 μM, 100 μM), an MAPKK inhibitor, to analyzeNox1 expression in the K-Ras-NRK cells and serum- and EGF-induced Nox1expression by real-time quantitative polymerase chain reaction.

As a result, Nox1 expression in the K-Ras-NRK cells was inhibited by thePD98059 concentration-dependently (FIG. 1(a)). Moreover, serum- andEGF-induced Nox1 expression was respectively inhibited by 20 μM PD98059(FIGS. 1(c) and (d)).

On the other hand, wortmannin (100 nM), a PI3 kinase inhibitor, did notinhibit an increase in Nox1 expression in the H-Ras-NIH3T3 cells in acontrol experiment (data not shown). These results indicated that thesignals of mutant Ras gene and growth factors thereof induce Nox1expression, by pathways not via PI3K but via Ras-MAPKK-MAPK.

EXAMPLE 4 Involvement of Nox1 Gene in Transformation with Mutant RasGene

Oligonucleotides 1-S and 1-AS were annealed and subcloned into apSilencer hygro vector with an H1-promoter (Ambion) to yield RNAi(1).Likewise, oligonucleotides 3-S and 3-AS were annealed and subcloned intoa pSilencer hygro vector with an H1-promoter (Ambion) to yield RNAi(3).In addition, oligonucleotides 5-S and 5-AS were annealed and subclonedinto a pSilencer hygro vector with an H1-promoter (Ambion) to yieldRNAi(5). A pSilencer hygro Negative Control plasmid (Ambion) was used asa control vector.

The RNAi(1) is an siRNA construction targeted for positions from 223 to241 of SEQ ID NO: 3. The RNAi(3) is an siRNA construction targeted forpositions from 578 to 596 of SEQ ID NO: 3. The RNAi(5) is an siRNAconstruction targeted for positions from 1224 to 1242 of SEQ ID NO: 3.

The sequence of each oligonucleotide is as follows: 1-S:5′-GATCCCGTTATGAGAAGTCTGACAAGTTCAA (SEQ ID NO: 18)GAGACTTGTCAGACTTCTCATAATTTTTTGGAA A-3′ 1-AS:5′-AGCTTTTCCAAAAAATTATGAGAAGTCTGAC (SEQ ID NO: 19)AAGTCTCTTGAACTTGTCAGACTTCTCATAACG G-3′ 3-S:5′-GATCCCGATTCTTGGCTAAATCCCATTCAAG (SEQ ID NO: 20)AGATGGGATTTAGCCAAGAATCTTTTTTGGAA A-3′ 3-AS:5′-AGCTTTTCCAAAAAAGATTCTTGGCTAAATC (SEQ ID NO: 21)CCATCTCTTGAATGGGATTTAGCCAAGAATCG G-3′ 5-S:5′-GATCCCGGACATTTGAACAACAGCATTCAAG (SEQ ID NO: 22)AGATGCTGTTGTTCAAATGTCCTTTTTTGGAA A-3′ 5-AS:5′-AGCTTTTCCAAAAAAGGACATTTGAACAACA (SEQ ID NO: 23)GCATCTCTTGAATGCTGTTGTTCAAATGTCCG G-3′

4 μg each of the RNAi(1), RNAi(3), RNAi(5), and pSilencer hygro NegativeControl plasmid was transfected respectively into 1×10⁶ K-Ras-NRK cellsusing Lipofectamine (Gibco-BRL). The transfected cells were cultured forselection at 37° C. for 2 to 3 weeks in a DMEM medium supplemented with10% fetal bovine serum (FBS) and 400 μg/ml hygromycin B under a 5% CO₂moisture environment. After the culture, surviving colonies wereisolated.

Three clones of the cell lines stably transfected with each of theRNAi(1), RNAi(3), and RNAi(5) (K-Ras-NRK/RNAi(1)-7,K-Ras-NRK/RNAi(1)-12, and K-Ras-NRK/RNAi(1)-15; K-Ras-NRK/RNAi(3)-17,K-Ras-NRK/RNAi(3)-19, and K-Ras-NRK/RNAi(3)-96; and K-Ras-NRK/RNAi(5)-2,K-Ras-NRK/RNAi(5)-3, and K-Ras-NRK/RNAi(5)-7) as well as two clones ofthe cells stably transfected with the pSilencer hygro Negative Controlplasmid (K-Ras-NRK/neg-1 and K-Ras-NRK/neg-2) were selected. Thetransfection of these constructions was confirmed by PCR using M13F and3.0Rev as primers (FIG. 2). PCR temperature conditions were set to 94°C. for 2 minutes, 30 cycles of (94° C. for 1 minute, 60° C. for 1minute, and 72° C. for 1 minute), followed by 72° C. for 10 minutes. Athermal cycler used was Takara Thermal Cycler SP (Takara Shuzo CompanyLimited). (Primer Sequence) (SEQ ID NO: 24) M13F:5′-GTTTTCCCAGTCACGAC-3′ (SEQ ID NO: 25) 3.0Rev:5′-GAGTTAGCTCACTCATTAGGC-3′

Next, celluler morphological changes in which Nox1 gene was involvedwere compared among the cells. As a result, as shown in FIG. 3, theK-Ras-NRK cells transfected with the RNAi(1) to RNAi(3) were extended,whereas the K-Ras-NRK cells transfected with the pSilencer hygroNegative Control plasmid morphologically remained round as the cellsobserved before introduction.

Subsequently, soft agar culture assay was performed in order to examinethe transformation of the cells by observing anchorage-independentgrowth capacity. An agarose layer containing 0.53% agar and nutrientsnecessary for cell growth was placed and solidified in a culture dish of6 cm in diameter, on which the cells suspended in a DMEM containing 0.3%agar and 10% FBS were then piled up at a final concentration of 1.5×10⁴cells/culture dish. Subsequently, the cells were cultured at 37° C.under a 5% CO₂ moisture environment to observe the appearance of cellcolonies over 3 weeks. The adhesion-dependent cell growth of the celllines exhibited by the soft agar culture was measured, and +/−averagestandard deviation (s.e.m.) from three measurements was shown in FIG. 4.As a result, the formation of many colonies was observed in theK-Ras-NRK cells and the transfected cells with the pSilencer hygroNegative Control plasmid (neg-1 and neg-2), whereas colony formation wasremarkably inhibited in the transfection with the RNAi(1) to RNAi(5).

In addition, 10⁴ cells each of the K-Ras-NRK/neg-1 (neg-1),K-Ras-NRK/RNAi(1)-7 (i(1)-7), K-Ras-NRK/RNAi(3)-19 (i(3)-19), andK-Ras-NRK/i(5)-7 (i(5)-7) cells were transferred from the culture dishto a culture solution and then liquid-cultured for 6 days in a standardculture solution (DMEM containing 10% FBS) in the presence of 5% CO₂.Subsequently, the number of the cells in the liquid medium was measured,and its growth curve was shown in FIG. 5. As a result, each growth rateof the K-Ras-NRK/RNAi(1)-7, K-Ras-NRK/RNAi(3)-19, andK-Ras-NRK/RNAi(5)-7 cells was also decreased in the liquid culture, andby contrast, inhibitory effect on growth was not observed in theK-Ras-NRK/neg-1.

EXAMPLE 5 Confirmation of Superoxide Production by NADPH Oxidase Codedfor Nox1 Gene

The present inventors measured superoxide production by NBT reductionanalysis using the transformed cells of Example 4 in order to evaluatethe role of Nox1 in superoxide production induced by mutant Ras. In thepresent example, the method of Suh et al. was used in the NBT (nitrobluetetrazolium) analysis (1). Namely, 0.2 ml of a Hanks solution containing0.25% NBT was prepared with or without 40 units of superoxide dismutase(SOD), a reactive oxygen-digesting enzyme, in which 2×10⁵ cells were inturn suspended and treated at 37° C. for 8 minutes. The treated cellswere separated by low-speed centrifugation and solubilized by theaddition of 0.5 ml of pyridine. Absorbance at 510 nm was measured toquantify a decrease in NBT (extinction coefficient of 11,000/M/cm). Theobtained data was shown in FIG. 6 with +/−s.e.m. from 3 measurements.

As a result of the analysis, the K-Ras-NRK and K-Ras-NRK/neg-1 increasedNBT reduction, which was inhibited by the superoxide dismutasetreatment, as compared with the NRK. By contrast, theK-Ras-NRK/RNAi(1)-7, K-Ras-NRK/RNAi(3)-19, and K-Ras-NRK/RNAi(5)-7decreased the ROS production-stimulating effect of K-Ras oncogene to thelevel of the NRK cells (FIG. 6), thereby indicating that Nox1 isinvolved in an increase in superoxide production in mutantRas-transformed cells.

EXAMPLE 6 Confirmation of Nox1 Expression Decreased by RNAi(1), RNAi(3),and RNAi(5)

The present inventors conducted analysis in the following procedures inorder to confirm Nox1 expression decreased by the RNAi(1), RNAi(3), andRNAi(5): GFP-rat Nox1 was subjected to cotransfection with each of theRNAi(1) to (5) and subsequently immunoblotting analysis was conducted inthe same way as in Example 2 in order to evaluate the inhibitory effectof the RNAi(1), RNAi(3), and RNAi(5) on GFP-rat Nox1 expression. Asshown in the left panel of FIG. 7, it was revealed that an expectedGFP-rat Nox1 fusion polypeptide is produced, and that the coexpressionof the RNAi(1), RNAi (3), and RNAi(5) suppresses the production of aGFP-rat Nox1 fusion protein dependently on a dose of these vectors.GFP-Nox1 expression shown in the left panel of FIG. 7 was quantitativelydetermined by immunoblotting using an anti-GFP antibody. Numerals in theleft panel of FIG. 7 denote the amount (μg) of DNA transfected. BothRNAi(1) and RNAi(5) are targeted for rat Nox1. The same approach as inthe left panel of FIG. 7 was used to show in the right panel of FIG. 7that the RNAi(1) and RNAi(5) do not inhibit human Nox1, that is, theRNAi constructions have high specificity to their target gene.

Moreover, RT-PCR demonstrated that the mRNA expression of endogenousNox1 is certainly inhibited in each of the K-Ras-NRK/RNAi(1)-7,K-Ras-NRK/RNAi(1)-12, K-Ras-NRK/RNAi(3)-19, K-Ras-NRK/RNAi(3)-96,K-Ras-NRK/RNAi(5)-2, K-Ras-NRK/RNAi(5)-7 cells, as compared to theK-Ras-NRK and K-Ras-NRK/neg-1 cells. The result is shown in FIG. 8.

Sequences of PCR Primers used in Example 6 (SEQ ID NO: 26) ForwardPrimer: 5′-ATGGGAAACTGGCTGGTTA-3′ (SEQ ID NO: 27) Reverse Primer:5′-TCAGAACGTTTCTTTGTTGAA-3′

The results of the present Example indicate that an increase in theexpression rate of Nox1 gene and transformation with mutant Ras areassociated with each other, as well as morphological changes caused bythe constitutional changes of cytoskeletons and adhesive proteins.

EXAMPLE 7 Influence of Reactivation of Nox1 Once Inhibited by siRNA onCell

pEGFP-C1 (GFP) and pEGFP-human Nox1 (GFP-Nox1) were separatelytransfected into the K-Ras-NRK/RNAi(1)-7 and K-Ras-NRK/RNAi(5)-7 by thesame approach as in Example 4, then immunoblotting was conducted by thesame approach as in Example 6 (FIG. 9). As shown in Example 6 and theright panel of FIG. 7, neither RNAi(1) nor RNAi(5) inhibits human Nox1expression. Therefore, even if the pEGFP-human Nox1 is transfected intothe K-Ras-NRK/RNAi(1)-7 and K-Ras-NRK/RNAi(5)-7 cells, this human Nox1is not inhibited by siRNA.

As shown in FIG. 10, the K-Ras-NRK/RNAi(1)-7 cells could not derepresssiRNA and were oblate only by introducing the control vector therein(i(1)-7+GFP), whereas they morphologically returned to the sphericalshape close to the original K-Ras-NRK cells by transfecting thepEGFP-human Nox1 therein (i(1)-7+GFP-Nox1-3) (FIG. 10).

Growth curves obtained by the same approach as in Example 4 alsoexhibited accelerated growth in the pEGFP-human Nox1-incorporatedK-Ras-NRK/RNAi(1)-7 (GFP-Nox1-3) and pEGFP-human Nox1-incorporatedK-Ras-NRK/RNAi(5)-7 (GFP-Nox1-1), as compared with the controlvector-incorporated K-Ras-NRK/RNAi(1)-7 (GFP-59) and the controlvector-incorporated K-Ras-NRK/RNAi(5)-7 (GFP-78) (FIG. 11). Moreover,the soft agar culture assay in the same way as in Example 4 alsodemonstrated that colony formation in the pEGFP-human Nox1-incorporatedK-Ras-NRK/RNAi(1)-7 (GFP-Nox1-2 and GFP-Nox1-3) recovered to the samelevel as that in the original K-Ras-NRK cells, as compared with thecontrol vector-incorporated K-Ras-NRK/RNAi(1)-7 (i(1)-7+GFP-59 andi(1)-7+GFP-60) (FIG. 12).

EXAMPLE 8 Influence of Nox1 Gene Expression on Transformation withMutant Ras

Diphenylene iodonium (DPI), a Nox1 inhibitor, was used to examine theinfluence of Nox1 gene expression on the morphologies of the cellstransformed with mutant Ras. Namely, K-Ras-NRK cells and NRK cells werecultured overnight at 37° C. in a medium containing 20 μM DPI or 30 μMPD98059 under a 5% CO₂-containing moisture environment to observemorphological changes. Photographs documenting the cell morphologies areshown in FIG. 13.

As shown in FIG. 13, the K-Ras-NRK cells transiently assumed a flatshape that was close to the shape of the NRK cells, when the K-Ras-NRKcells were treated overnight with flavoprotein inhibitor DPI or 10 mMantioxidant n-acetyl cysteine (data not shown). As further shown in FIG.13, such morphological changes were also observed in the K-Ras-NRK cellstreated with the PD98059. Thus, these results indicated that an increasein Nox1 gene expression is essential to Ras-MAPKK-MAPK pathway-promotedtransformation with mutant Ras.

EXAMPLE 9 Influence of siRNA on Tumor Formation Caused by Mutant Ras

The K-Ras-NRK/neg-1, K-Ras-NRK/RNAi(1)-7, K-Ras-NRK/RNAi(1)-12,K-Ras-NRK/RNAi(3)-19, K-Ras-NRK/RNAi(3)-96, and K-Ras-NRK/RNAi(5)-2cells were separately transplanted into athymic mice to observe tumorformation. Namely, 10⁶ cells each of the cells were suspended in 0.2 mlPBS and then subcutaneously transplanted into the nude mice (athymic:Nu/Nu). Then, tumor formation in all of the nude mice was observed over1 month, while the volume of the formed tumor was measured. The resultwas shown in FIG. 14. In FIG. 14, the solidly shaded bars denote thevolume of tumor; the error bars denote s.e.m.; and the fractions denotethe proportion of tumor-bearing mice to all the mice used (tumor/total).

As shown in FIG. 14, the K-Ras-NRK/neg-1 formed active tumor within 2weeks. By contrast, the K-Ras-NRK/RNAi(1)-7, K-Ras-NRK/RNAi(1)-12,K-Ras-NRK/RNAi(3)-19, and K-Ras-NRK/RNAi(3)-96 remarkably suppressedtumor growth. When histologically observed by anatomy, the tumor formedin the K-Ras-NRK/neg-1 resulted in increased blood vessels. This isconsistent with the possibility that Nox1 gene induces angiogenesiscaused by increase in vascular endothelial growth factor production (1).The small tumor formed in the K-Ras-NRK/RNAi(1)-7, K-Ras-NRK/RNAi(1)-12,K-Ras-NRK/RNAi(3)-19, and K-Ras-NRK/RNAi(3)-96 exhibited signs ofnecrosis in most of the cells (data not shown).

As seen from these experimental results, mutant Ras (oncogene) elevatesmitogenic oxidase Nox1 via MAPKK-MAPK pathways, whereas Nox1 geneexpression is essential to transformation of cells, cancer formation,and its progression caused by the Ras. Namely, the studies conducted bythe present inventors revealed a molecular mechanism in which a Nox1polypeptide plays an essential role as a redox signal molecule intransformation with mutant Ras.

Nox family proteins Nox1 to 5, Gp91phox homologues, were identified fromnon-phagocytic cells and reported to respectively have specificfunctions in various cell processes (1 and 5 to 9). Among the Noxfamily, the Nox1 gene is unique in that it codes for transmembraneoxidase that mediates the mitogenic signals from growth factors (1 and4) and oncogenes.

Considering the involvement of ROS in the promotion of tumor growth andthe possibility of ROS production for inducing ROS-producing enzymes inmalignant cancer cells (7), Nox1 gene and its polypeptides are probablycapable of suppressing cancer progression and serving as targetmolecules that allow the cancer therapy.

The SEQ ID NOs in the sequence listing of the present specificationrepresent the following sequences:

SEQ ID NO: 1 represents the nucleotide sequence of mRNA transcribed froma human Nox1 gene or cDNA;

SEQ ID NO: 2 represents the amino acid sequence of a polypeptide codedby the human Nox1 gene;

SEQ ID NO: 3 represents the nucleotide sequence of mRNA transcribed froma rat Nox1 gene or cDNA;

SEQ ID NO: 4 represents the amino acid sequence of a polypeptide codedby the rat Nox1 gene;

SEQ ID NO: 5 represents the nucleotide sequence of a forward primercorresponding to a Nox1 gene used in a diagnostic method for humancancer;

SEQ ID NO: 6 represents the nucleotide sequence of a reverse primercorresponding to a Nox1 gene used in a diagnostic method for humancancer;

SEQ ID NO: 7 represents the sequence of a forward primer used in thedetection of a human Nox1 gene by real-time quantitative polymerasechain reaction;

SEQ ID NO: 8 represents the sequence of a reverse primer used in thedetection of a human Nox1 gene by real-time quantitative polymerasechain reaction;

SEQ ID NO: 9 represents the sequence of a TaqMan probe used in thedetection of a human Nox1 gene by real-time quantitative polymerasechain reaction;

SEQ ID NO: 10 represents the nucleotide sequence of siRNA for a humanNox1 gene of the present invention;

SEQ ID NO: 11 represents the nucleotide sequence of siRNA for a humanNox1 gene of the present invention;

SEQ ID NO: 12 represents the nucleotide sequence of siRNA for a rat Nox1gene of the present invention;

SEQ ID NO: 13 represents the nucleotide sequence of siRNA for a rat Nox1gene of the present invention;

SEQ ID NO: 14 represents the nucleotide sequence of siRNA for a rat Nox1gene of the present invention;

SEQ ID NO: 15 represents the nucleotide sequence of a forward primerused in Example 1 for detecting the presence or absence of Nox1expression by real-time quantitative polymerase chain reaction;

SEQ ID NO: 16 represents the nucleotide sequence of a reverse primerused in Example 1 for detecting the presence or absence of Nox1expression by real-time quantitative polymerase chain reaction;

SEQ ID NO: 17 represents the nucleotide sequence of a TaqMan MGB probeused in Example 1 for detecting the presence or absence of Nox1expression by real-time quantitative polymerase chain reaction;

SEQ ID NO: 18 represents the nucleotide sequence of an oligonucleotidethat constitutes an siRNA construction used in Example 4 for examiningthe involvement of a Nox1 gene in transformation with a mutant Ras gene;

SEQ ID NO: 19 represents the nucleotide sequence of an oligonucleotidethat constitutes an siRNA construction used in Example 4 for examiningthe involvement of a Nox1 gene in transformation with a mutant Ras gene;

SEQ ID NO: 20 represents the nucleotide sequence of an oligonucleotidethat constitutes an siRNA construction used in Example 4 for examiningthe involvement of a Nox1 gene in transformation with a mutant Ras gene;

SEQ ID NO: 21 represents the nucleotide sequence of an oligonucleotidethat constitutes an siRNA construction used in Example 4 for examiningthe involvement of a Nox1 gene in transformation with a mutant Ras gene;

SEQ ID NO: 22 represents the nucleotide sequence of an oligonucleotidethat constitutes an siRNA construction used in Example 4 for examiningthe involvement of a Nox1 gene in transformation with a mutant Ras gene;

SEQ ID NO: 23 represents the nucleotide sequence of an oligonucleotidethat constitutes an siRNA construction used in Example 4 for examiningthe involvement of a Nox1 gene in transformation with a mutant Ras gene;

SEQ ID NO: 24 represents the nucleotide sequence of an M13 primer usedin Example 4 for confirming the transfection of the siRNA constructionsfor a Nox1 gene;

SEQ ID NO: 25 represents the nucleotide sequence of a 3.0 Rev primerused in Example 4 for confirming the transfection of the siRNAconstructions for a Nox1 gene;

SEQ ID NO: 26 represents the nucleotide sequence of a forward primerused in Example 6 for confirming a decrease in Nox1 expression; and

SEQ ID NO: 27 represents the nucleotide sequence of a reverse primerused in Example 6 for confirming a decrease in Nox1 expression.

REFERENCES

-   1. Suh, Y-A, et al. Nature 401, 79-82 (1999)-   2. Arnold, R. S. et al. Proc. Natl. Acad. Sci. USA. 98, 5550-5555    (2001)-   3. Arbiser, J. L. et al. Proc. Natl. Acad. Sci. USA. 99, 715-720    (2001)-   4. Lassegue B. et al. Circ. Res. 88, 888-894 (2001)-   5. Lambeth, J. D., Dheng, G., Arnold, R. S. & Edens, W. A. TIBS. 25,    459-461 (2000)-   6. Royer-Pokora, B. et al. Nature 322, 32-38 (1986)-   7. Kikuchi, H., Hikage, M., Miyashita, H. & Fulumoto, M. Gene 269,    131-140 (2001)

1. A composition for producing an antibody, comprising: (1) apolypeptide comprising the amino acid sequence of SEQ ID NO: 2; (2) apolypeptide having an amino acid sequence mutated from the amino acidsequence of SEQ ID NO: 2 by the substitution, deletion, addition, and/orinsertion of one or more amino acid residues and inducing the productionof an antibody specific to the polypeptide comprising the amino acidsequence of SEQ ID NO: 2; or (3) a polypeptide fragment having a partialsequence of the polypeptide of (1) or (2) and inducing the production ofan antibody specific to the polypeptide comprising the amino acidsequence of SEQ ID NO:
 2. 2. The composition according to claim 1,wherein the antibody is an antibody for detecting a human cancer cell.3. The composition according to claim 1, comprising a polypeptidecomprising the amino acid sequence of SEQ ID NO:
 2. 4. The compositionaccording to claim 1, comprising a polypeptide having a partial sequenceof the amino acid sequence of SEQ ID NO: 2 and inducing the productionof an antibody specific to the polypeptide comprising the amino acidsequence of SEQ ID NO:
 2. 5. The composition according to claim 1,comprising a polypeptide fragment having a partial sequence of the aminoacid sequence of SEQ ID NO: 2 and inducing the production of an antibodyspecific to the polypeptide comprising the amino acid sequence of SEQ IDNO:
 2. 6. A method for producing an antibody specific to a polypeptidecomprising the amino acid sequence of SEQ ID NO: 2, comprisingadministering a composition according to claim 1 to a mammal.
 7. Anantibody specific to a polypeptide comprising the amino acid sequence ofSEQ ID NO:
 2. 8. The antibody according to claim 7, wherein the antibodyis a human/mouse chimeric antibody, a humanized antibody, or a humanantibody.
 9. The antibody according to claim 7, wherein the antibody isa polyclonal antibody or a monoclonal antibody.
 10. A diagnostic methodfor cancer, comprising bringing an antibody according to claim 7 intocontact with a biological sample.
 11. A diagnostic kit for cancer,comprising an antibody according to claim
 7. 12. A pharmaceuticalcomposition for cancer therapy, comprising an antibody according toclaim
 7. 13. The pharmaceutical composition for cancer therapy accordingto claim 12, wherein the antibody is a human/mouse chimeric antibody, ahumanized antibody, or a human antibody.
 14. The pharmaceuticalcomposition for cancer therapy according to claim 12, wherein theantibody is a polyclonal antibody or a monoclonal antibody.
 15. Thepharmaceutical composition for cancer therapy according to claim 12,further comprising an appropriate carrier.
 16. A diagnostic method forcancer, characterized by detecting a polynucleotide comprising thenucleotide sequence of SEQ ID NO: 1 or a fragment thereof.
 17. Thediagnostic method for cancer according to claim 16, wherein thepolynucleotide or the fragment thereof is detected by polymerase chainreaction (PCR) or real-time quantitative polymerase chain reaction. 18.The diagnostic method for cancer according to claim 17, wherein thedetection is performed by PCR using a sense strand fragmentcorresponding to the nucleotide sequence of SEQ ID NO: 1 as a forwardprimer and an antisense strand fragment corresponding to the nucleotidesequence of SEQ ID NO: 1 as a reverse primer.
 19. The diagnostic methodfor cancer according to claim 18, wherein the forward primer has 14 to60 bases in length and the reverse primer has 14 to 60 bases in length.20. The diagnostic method for cancer according to claim 18, wherein thediagnostic method uses the following forward and reverse primers: (SEQID NO: 5) the forward primer of 5′-GGAGCAGGAATTGGGGTCAC-3′; and (SEQ IDNO: 6) the reverse primer of 5′-TTGCTGTCCCATCCGGTGAG-3′.


21. The diagnostic method for cancer according to claim 17, wherein thedetection is performed by real-time quantitative polymerase chainreaction using a sense strand fragment corresponding to the nucleotidesequence of SEQ ID NO: 1 as a forward primer and an antisense strandfragment corresponding to the nucleotide sequence of SEQ ID NO: 1 as areverse primer.
 22. The diagnostic method for cancer according to claim21, wherein the diagnostic method uses the following forward and reverseprimers and TaqMan probe: the forward primer of (SEQ ID NO: 7)5′-CCACTGTAGGCGCCCTAAGTT-3′; the reverse primer of (SEQ ID NO: 8)5′-AAGAATGACCGGTGCAAGGA-3′; and the TaqMan probe of (SEQ ID NO: 9)5′-AAGGGCATCCCCCTGAGTCTTGGAA-3′.


23. siRNA corresponding to a polynucleotide comprising the nucleotidesequence of SEQ ID NO: 1 or a fragment thereof.
 24. The siRNA accordingto claim 23, corresponding to a polynucleotide comprising a nucleotidesequence at positions from 71 to 1615 of SEQ ID NO: 1 or a fragmentthereof.
 25. The siRNA according to claim 23, wherein the siRNA has anucleotide sequence from 8 to 30 bp in length.
 26. The siRNA accordingto claim 23, wherein the siRNA consists of a nucleotide sequenceselected from the group consisting of the following nucleotide sequencesof SEQ ID NOs:10 to 14: (SEQ ID NO: 10)5′-GCGTGGCTTCAGCATGGAATTCAAGAGATTCCATGCTGAAGCCACGC TTTTTTGGAAA-3′; (SEQID NO: 11) 5′-GGGCTTTCGAACAACAATATTCAAGAGATATTGTTGTTCGAAAAGCCCTTTTTTGGAAA-3′; (SEQ ID NO: 12)5′-GTTATGAGAAGTCTGACAAGTTCAAGAGACTTGTCAGACTTCTCATA ATTTTTTGGAAA-3′; (SEQID NO: 13) 5′-GATTCTTGGCTAAATCCCATTCAAGAGATGGGATTTAGCCAAGAATCTTTTTTGGAAA-3′; and (SEQ ID NO: 14)5′-GGACATTTGAACAACAGCATTCAAGAGATGCTGTTGTTCAAATGTCC TTTTTTGGAAA-3′.


27. A pharmaceutical composition for cancer therapy, comprising: siRNAcorresponding to a polynucleotide comprising the nucleotide sequence ofSEQ ID NO: 1 or a fragment thereof; and siRNA corresponding to apolynucleotide comprising a nucleotide sequence at positions from 71 to1615 of SEQ ID NO: 1 or a fragment thereof.
 28. A pharmaceuticalcomposition for cancer therapy, comprising a cell transformed with siRNAcorresponding to a polynucleotide comprising the nucleotide sequence ofSEQ ID NO: 1 or a fragment thereof; and siRNA corresponding to apolynucleotide comprising a nucleotide sequence at positions from 71 to1615 of SEQ ID NO: 1 or a fragment thereof.
 29. The pharmaceuticalcomposition for cancer therapy according to claim 28, wherein the cellto be transformed is a cell taken out of a patient to be treated.
 30. Amethod for producing a cell for cancer therapy, comprising preparing ahuman cell and transforming the cell with siRNA corresponding to apolynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or afragment thereof; and siRNA corresponding to a polynucleotide comprisinga nucleotide sequence at positions from 71 to 1615 of SEQ ID NO: 1 or afragment thereof.
 31. The method for producing a cell for cancer therapyaccording to claim 30, wherein the human cell is a cell taken out of apatient to be treated.
 32. A screening method for a cancer cell growthinhibitor targeted for a Nox1 gene, comprising: transfecting a cellhaving a mutant Ras gene with a Nox1 gene; bringing the transformed cellinto contact with a substance to be screened; and detecting theexpression of the Nox1 gene and the inactivation of Nox1 activity. 33.The screening method according to claim 32, comprising culturing thetransformed cell together with the substance to be screened.
 34. Thescreening method according to claim 32, wherein the expression of theNox1 gene is detected by detecting mRNA by real-time quantitativepolymerase chain reaction or detecting a polypeptide or a peptidefragment thereof coded by the Nox1 gene with an antibody.
 35. Thescreening method according to claim 32, wherein the expression of theNox1 gene is detected by observing morphological changes in thetransformed cell.
 36. The screening method according to claim 32,wherein the cell having a mutant Ras gene is an H-Ras-NIH3T3 cell or aK-Ras-NRK cell.
 37. The screening method according to claim 32, whereinthe transfection of the Nox1 gene is performed using pEGFP-C1(K-Ras-NRK/GFP) or pEGFP-C1-Nox1 (K-Ras-NRK/GFP-Nox1).
 38. A cell havinga mutant Ras gene, which is transfected with a Nox1 gene.